Diabetes self-care activities, like, healthy diet, regular exercise, self-monitoring of blood glucose, and rational use of medicines are considered to play a vital role in establishing euglycemia. Health literacy among type 2 diabetes mellitus (T2DM) patients in Pakistan is very low, which is the most likely cause for poor clinical outcomes. This study is designed to investigate the impact of pharmacist-led educational intervention on glycemic control, self-care activities and disease knowledge among T2DM patients in Pakistan.In this randomized controlled trail, effectiveness of a 6-month pharmacist-led educational intervention will be examined on glycemic control, diabetes self-care activities and disease knowledge of 80 adult T2DM patients (age >30 years) with poorly controlled T2DM (HbA1c> 7%), after randomizing them into intervention and control groups, at diabetes care clinic of Capital Hospital Islamabad, Pakistan.The primary outcome is change in patients’ HbA1c, whereas, changes in self-care activities and patients’ disease knowledge are the secondary outcomes. After baseline assessment of their self-care activities and disease knowledge by using validated Urdu versions of Diabetes Self-management Questionnaire (DSMQ) and Diabetes Knowledge Questionnaire (DKQ), respectively, interventional group patients will be supplemented with a face-to-face pharmacist-led educational intervention, whereas, the control group will receive usual care. Intervention arm patients will be educated successively at their first follow-up visit (12th week) and telephonically after every 4 weeks. All assessments will be made at baseline and end of trail for both intervention and control groups. Multivariate general linear model will be applied to analyze the effects of the intervention.Glycemic control in T2DM patients requires optimum self-care activities. This study is an attempt to improve self-care behaviors among poorly controlled T2DM patients who are at higher risk of diabetes-associated late complications. According to World Health Organization (WHO), the diabetes number increased from 108 million in 1980 to 422 million in 2014.–8 According to the current statistics of International Diabetes Federation (IDF), in Pakistan there are 7.028 million cases of adults (20–79 years) with diabetes, with 6.9% prevalence rate and 2.928 million undiagnosed adults with diabetes.Pakistan has been ranked 7th in diabetes disease burden in the world, it is projected to reach 15% by 2030, and if the present scenario continues, Pakistan is expected to move to top 4th place. requiring patients to adopt specific life style modifications in addition to continued pharmacotherapy. Because of the chronic nature of T2DM, diabetes self-care activities have become an integral component of effective diabetes care around the globe. Glycemic control, measured in terms of glycosylated hemoglobin (HbA1c), has been associated with significant reduction in diabetes-related complications and economic burden.T2DM is the most prevalent (>90%) type of diabetes,T2DM is a self-managed, chronic metabolic disorder, where patients has to take major responsibility to manage their condition by adopting healthy self-care behaviors. The consequences of these self-care behaviors can directly affect health. Managing T2DM requires a huge amount of time, modifications in lifestyle, and confidence to do this. Because of the high cost associated with controlling diabetes, health care providers are now taking an active role in the provision education for diabetes self-management.–16 Structured self-management education programmes can be delivered on a one-to-one basis or to groups of people with diabetes and should be facilitated by trained educators. The aim of the education is not only to improve knowledge and skills, but also to motivate a person to make and sustain lifestyle changes, by giving them the confidence to make their own treatment/lifestyle choices on a day-to-day basis.Educational interventions can play an important role to improve patients’ knowledge and skills regarding diabetes self-management. Systematic reviews of interventions addressing diabetes self-management indicate that the diabetes education courses enhance knowledge, self-care behaviors, and reduction in HbA1C. Health literacy has been reported as a significant factor influencing the patients’ glycemic control. Clinical outcome can be achieved by addressing patients’ literacy. Pharmacist-based educational interventions are associated with improved medication adherence and glycemic control.–21 A review of 16 pharmacist-based interventions resulted in an absolute reduction of HbA1c with an average of 0.62%. Similarly, in an another review, a reduction in A1c levels up to 2.1% was demonstrated by 20 pharmacist-based interventions. A study examining the effectiveness of a free rural clinic for diabetes patients managed by pharmacists found that patients who received education on diabetes, lifestyle modifications, and management of diabetes drug therapy had significant reductions in A1c (≥ 1%) after 24 months.Educational intervention on self-management can be delivered as group education or individually as one-to-one approach. Sturt et al. reported that an individualized approach with the help of a diabetes guide and telephonic support has demonstrated clinically significant reductions in A1c levels, for T2DM patients with poor glycemic control.Keeping in view the importance educational interventions in diabetes self-care and clinical outcomes, we designed a face-to-face short-term (6 months) pharmacist-led educational intervention model in poorly controlled T2DM patient (HbA1c >7%) in Pakistan. In this study, we will investigate the mean change in HbA1C at 6 months after the baseline, and a comparison will be made with a similar comparator group of T2DM patients receiving standard diabetes care. In addition, comparison between self-care activities and disease knowledge between the groups will also be studied.22.1This is a 24-week open label, parallel group, prospective randomized controlled trial. In this study, the impact of a pharmacist-based, face-to-face educational intervention will be investigated in adult Pakistani people with poorly controlled HbA1c >7%) type 2 diabetes. The study flow is presented in , located in Islamabad, Pakistan. The recruitment and data collection will be performed between October 2017 and March 2018. Setting for pharmacist educational intervention in this study will be a private counselling room at CDA diabetes outpatient clinic.2.3Eligibility criteria for the study includes poorly controlled (HbA1c>7%) T2DM, adult patients (age ≥ 30 years) regardless of gender; speak and understand Urdu and / or English languages; having no significant comorbidity; being not involved in any trial/study related to diabetes during last 3 months and able to attend regular visits. Participants will be excluded if they are of other types of diabetes ; unable to answer the questionnaire independently or having hearing, vision, or cognitive impairments.2.4Patients will be identified from the hospital record and who will meet the inclusion criteria of the study will be invited telephonically for participation in the study. If the patient is willing to participate in the study, an appointment will be set in the outpatient counselling room of the pharmacist at CDA hospital and purpose of the study will be explained to the patient and his/her written informed consent will be obtained and baseline assessments will be done. Simple random sampling technique will be used from a list of random numbers of eligible patients which will be compiled by using the patients’ hospital identification numbers. After recruitment, the patients will be requested to handpick an envelope from the basket indicating allocation to either control or intervention group with 1:1 randomization. AB will generate the random allocation sequences, MSN will enrol participants and HS will assign participants to intervention and control groups.2.52.5.1To investigate the effectiveness of a pharmacist-delivered diabetes management educational program on glycemic control (HbA1c) after completion of intervention (24th week).2.5.2To examine the impact of a pharmacist-delivered diabetes management educational program on self-care behaviors and patients’ diabetes knowledge, by using Urdu versions of 24-item Diabetes knowledge Questionnaire (DKQ) and 16-item Diabetes Self-management Questionnaire (DSMQ).2.5.3–28 for detecting the difference of 1% reduction for HbA1C (effect size) with standard deviation of 1.4%, at 6 months in the intervention group. A significance level of 0.05 is considered with study power of 80%. Based on these numbers, the calculated sample size is 62 ; however, a sample of 80 patients is assumed to be sufficient to compensate for a 25% attrition rate.Study sample size is calculated based on previous studies 2.5.4The participants will not be informed of their groups. After the initial assessment at baseline, the control group will receive usual care, whereas the interventional group will be provided with the structured intervention. A qualified pharmacist (researcher) will educate the patients in the interventional group about disease (diabetes), its symptoms, clinical goals, self-care activities , and reducing risks, whereas the control group patients (N = 40) will receive the usual care.The pharmacist will provide face-to-face diabetes management education (approximately 30 minutes duration) to the patients in the intervention group at their first visit, follow-up visit 12th week), and 4 telephone  and will recommend physician visits when necessary. In addition to face-to-face educational intervention patients in the intervention group will also be provided informatory brochures about self-care activities, and a log book to keep record of their self-monitored blood glucose levels. The educational intervention and patient informatory material has been designed according to American diabetes Association (ADA) guidelines, the details of are presented in Table 2th week,Details about patients’ (intervention and control group) demographics and lab profile  will be obtained by the pharmacist at first visit. At baseline and end of the trail 24th week), the diabetes disease knowledge and self-care activities of the patients in both groups (intervention and control) will be assessed by using 2 prevalidated questionnaires in Urdu language [24-item Urdu version of DKQ and 16-item Urdu version of DSMQ]. Pharmacists can assist the patient in completing questionnaires, but will not provide any help in answering the questions, as the same questionnaires will be used repeatedly. The details of study schedule for data collection are presented in Table 4th week,2.62.6.1 DKQ is a 24-item questionnaire. The DKQ scoring involves summing up the scores of all the correct items of each participant, where higher score will indicate better patient's diabetes knowledge. One point will be given to each correct answer and no point for the incorrect option.DKQ, developed for the Starr County Diabetes Education Study, has been translated and validated in Urdu language in Pakistan.2.6.2 Respondents will be asked to rate their specific self-care activities of last 3 months on a 16-item DSMQ. This questionnaire involves a four point likert scale (ranging from 0 – ‘does not apply to me’ to 3 – ‘applies to me very much’). There are 4 subscales of this study tool; namely, ‘Glucose Management’ (GM), which is comprised of 5 statements: 1, 4, 6, 10, 12, ‘Dietary Control’ (DC): comprised of 4 statements: 2, 5, 9, 13, ‘Physical Activity’ (PA): comprised of 3 statements: 8, 11, 15, and, ‘Health-Care Use’ (HU): comprised of 3 statements: 3, 7, 14. The last item (item 16) asks the respondents to rate their overall diabetes self-care, hence its score is included only in the ‘Sum Scale’ .DSMQ will be used to measure the patient's self-care activities. DMSQ was originally developed to evaluate diabetes self-care behaviors associated with glycemic control within common therapeutic options for adult patients of type 1 and type 2 DM.The scoring process of the DSMQ involves, adding up the scores of all 16 items after reversing the scores of 9 negatively keyed statements. Higher scores will represent more effective self-care. Finally DSMQ scores will be transformed to a scale ranging from 0 to 10, where a score of 10 will indicate the most effective self-care behavior.2.6.3t test will be used in case of normally distributed data, whereas, Mann–Whitney U test for non-normal distributions. Primary outcomes of the educational intervention, which are changes in HbA1C levels, self-care activities scores (DSMQ) and disease knowledge scores (DKQ) will be analyzed by using one-way analysis of variance (ANOVA) for normally distributed data and Kruskal Wallis Test for non-normally distributed data. P < 0.05 will be considered as significant for all analysis.The analyses will be performed using SPSS 22.0.0 . Frequencies and descriptive statistics will be used for patients’ demographic presentation, while means and standard deviations will be calculated for the continuous variables and group differences will be analyzed by using Pearson chi-square test for categorical variables. Kolmogorov-Smirnov test will be applied to check the distribution of data. Independent 2.6.4This study protocol has been approved by Human Research and Ethics committee of Capital Hospital, Islamabad, Pakistan, and, Australian New Zealand Clinical Trials Registry (Registration No. ACTRN12617001327370). Informed written consent will be obtained from all the study participants, after explaining them the purpose and procedure of the study. Participants can withdraw from the study willingly at any point of the study. Confidentiality of patients’ response and data will be assured. All procedures performed in this study involving human participants are in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. After completion of the trial a manuscript with detailed results will be published in a peer review journal.3–34 This article describes the study protocol of a RCT evaluating the effectiveness of a short educational intervention to improve self-care activities, disease knowledge and glycemic control in poorly controlled T2DM patients in Pakistan. In 2010, Qayyum et al. observed a clinically significant effect of diabetes self-management education (DSME) on HbA1c reduction in type 1 diabetes children in Pakistan. To date, pharmacist-led educational intervention has not been formally tested in T2DM patients in Pakistan. Therefore, this study will provide valuable information on the effectiveness of this intervention in terms of HbA1c reduction, improvements in disease knowledge and self-care activities.Previous trials of pharmacist-based educational interventions targeting self-management have demonstrated reductions in HbA1c levels in T2DM in various countries.This study protocol addresses the need to investigate the impact of new educational approaches to deliver a regular diabetes self-management support program. The research will also demonstrate the practicality of such educational intervention on a larger scale. We hypothesized that intervention arm patients will exhibit improvements in HbA1c, self-care activities and disease knowledge in comparison to the control group participants. Thus, we can assume that if 6 months intervention can produce significant reduction in HbA1c, it will also result in beneficial effect on patients’ long-term outcomes. A similar effect has also been reported in a recently published systematic review by Pousinho et al. (2016), where 24 studies out of 26 pharmacist-based interventions in the management of T2DM showed a greater reduction in HbA1c (difference ranging from -0.18% to -2.1%) in interventional group. Improvement in self-care activities has been reported in several pharmacist-based RCT studies.,33,36A systematic review by Wubben et al. (2008) on impact of pharmacist 18 interventional studies on diabetes patients demonstrated a significant reduction in HbA1c, ranging from +0.2 to -2.1% in intervention group as compared to control group.To date, limited studies have been conducted to address the association of self-care activities with glycemic control in T2DM patients in Pakistan. From a generalizability perspective, this study will recruit a diverse population across urban and rural Islamabad and Rawalpindi, and can be generalizable to other areas in Pakistan. If the study hypotheses are confirmed, the educational module and material could be applied in diabetes health care, resulting in reduction of diabetes related complications.3.1This interventional study is first of its kind to be conducted in Pakistan involving randomized control design. This study outcome will help to demonstrate the value of implementing a pharmacist-led educational intervention at diabetes care settings to improve self-care practices and clinical outcomes among Pakistani T2DM patients. A possible limitation of the study is the short duration (6 months intervention), which may not make it possible to examine intervention influence on diabetes related complications. Additionally, a 6-months intervention may result in high drop-out rate. To reduce the chances of drop-out, the researcher will facilitate patients in making appointments for follow-up assessment, and an additional 25% sample population will be recruited to compensate the drop-out.The authors thank the colleagues from the CDA hospital for their efforts in designing of this project. In addition, we would like to thank Global Asia 21 platform.4Conceptualization: A. Bukhsh, T.M. Khan.Data curation: A. Bukhsh, H. Sajjad, M.S. Nawaz.Formal analysis: A. Bukhsh, T.M. Khan.Investigation: A. Bukhsh, H. Sajjad, M.S. Nawaz.Methodology: A. Bukhsh, T.M. Khan.Project administration: A. Bukhsh, T.M. Khan.Supervision: A. Bukhsh, T.M. Khan.Validation: A. Bukhsh, H. Sajjad, T.M. Khan.Visualization: A. Bukhsh.Writing – original draft: A. Bukhsh.Writing – review & editing: T.M. Khan.


We prepared moth eye coatings on polymethylmethacrylate (PMMA) and polycarbonate using wafer-by-wafer step-and-flash nano-imprint lithography (NIL). The reduction in reflection in the visible field achieved with these coatings was 3.5% and 4.0%, respectively. The adhesion of the coating to both substrates was good. The moth eye coating on PMMA demonstrated good performance in three prototypical accelerated ageing tests. The pencil hardness of the moth eye coatings on both substrates was <4B, which is less than required for most applications and needs further optimization. Additionally, we developed a roll-to-roll UV NIL pilot scale process and produced moth eye coatings on polyethylene terephthalate (PET) at line speeds up to two meters per minute. The resulting coatings showed a good replication of the moth eye structures and, consequently, a lowering in reflection of the coated PET of 3.0%.Anti-reflective coatings (ARCs) are used to lower the reflection of light on the surface of a substrate. Here, we demonstrate that the two main drawbacks of moth eye-structured ARCs— The aectively ,7,8,9,10ectively . Exampleectively ,6,7 and Three main coating concepts exist to lower the reflection and increase the transmission of a substrate. Multilayer stacks with alternating thin layers of a high and low refractive index can be used to create an interference filter . They casn as the refractive index of the substrate and n0 as the refractive index of the medium [Secondly, an ARC can be realized using a coating of quarter wavelength thickness and a refractive index ofe medium ,15,16,17e medium ,15,16,17The third coating concept is so-called nanostructured graded-index coatings ,2. The nGraded-index, moth eye ARCs can be made using, e.g., nano-imprint lithography (NIL). NIL was developed in the mid-nineties as a technology for producing micro- and nano-structured surfaces ,31,32,33Currently, however, moth eye ARCs are not commonly used in large-scale applications. The main reasons for this are the lack of suitable liquid coating materials, which are qualified for this application, and the lack of a simple, low-cost, high-volume, large-scale application process. Most commercially available coating materials—(imprint) resists—are designed for subtractive lithography processes in which the resist is used for structuring silicon wafers and does not end up in the final product ,40,41,42This article shows experimental results demonstrating the potential to eliminate both disadvantages to a large extent. This enables cost-efficient and large-scale production of durable nanostructured moth eye-type ARCs on polymer substrates.To produce moth eye-type ARCs by full wafer step-and-flash NIL, we used the commercially available HT-AR-09 mold from Holotools. The mold was replicated in a hard polydimethylsiloxane (h-PDMS). Using the h-PDMS replicas, we made imprints in the commercially available UV-curable resist, Ormocomp, which has a refractive index of 1.535 at 500 nm and is available from Microresist Technology. This resist has been specifically designed for use in NIL. The imprints in the resist were made using the Nanoimprint Technology Platform from NIL Technology. The h-PDMS replica is pressed into the photosensitive coating material, which was applied using a roll bar, and cured with UV light through the replica. The approximate layer thickness of the Ormocomp coating is 15.4 µm. Finally, the h-PDMS replica is removed. The resulting coating layer has a moth eye structure identical to that of the HT-AR-09 mold. The residual layer thickness is about 15 µm. The residual resist layer can serve as a hard coat underneath the ARC. For regular single layer ARCs, the process involves two coating and curing steps: application of a transparent hard coat on the polymer substrate, curing of the hard coat, application of the ARC on the hard coat and curing of the ARC. Our process offers the potential to apply and cure the hard coat and top coat in one single step, which is a clear advantage over traditional single-layer ARCs on plastics.n = 1.49) made with these replicas are evaluated using Helium Ion Microscopy (HIM) and Atomic Force Microscopy (AFM). The results are displayed in The HT-AR-09 mold has the following specifications: the grating is a hexagonal array with a pitch of 250 nm, an average depth of 250 nm and a peak-to-peak distance of 300 nm. The surface structure of the h-PDMS replicas and the moth eye coatings on polymethylmethacrylate  in Reflection and transmission measurements of single-side coated substrates are performed under near normal angle for the visible wavelength regime (VIS) between 425 and 675 nm. The result for the moth eye-structured Ormocomp coating on PMMA is displayed in The average reflection of PMMA in the visible spectrum is 7.6%. Using the moth eye-structured Ormocomp coating on one side of the substrate, the reflection is lowered by 3.5%. As displayed in The average reflection for PC in the visible spectrum is 9.5%. The reflection is lowered by 4.0% after application of the moth eye-structured Ormocomp coating on one side of the substrate. As displayed in Both on PMMA and PC, the achieved anti-reflective effect matches the requirements for most commercial applications. The high degree of color neutrality of the coating matches the typical specification for application on displays.To evaluate the adhesion of the Ormocomp coating to PMMA and PC, scotch tape tests were performed according to ISO2409:1992. For that purpose, pressure sensitive tape was applied to an area of the coating, which was cross-hatched with scratched lines. Adhesion is considered adequate if the coating is not pulled off by the tape when it is removed. For the moth eye-structured Ormocomp coating on both substrates, no coating material was removed during the scotch tape test, indicating good adhesion.To evaluate the scratch resistance, pencil hardness tests according to ISO15184:1998 were performed for moth eye-structured and non-structured Ormocomp coatings on PC and PMMA. In all cases, three substrate samples were coated with Ormocomp resist, moth eye-structured using NIL and cured. Twenty-four hours after preparation, the pencil hardness of the coating was determined on three different positions per sample. Ergo, all values for pencil hardness are average values of nine measurements.The pencil hardness is expressed as follows: the first value is the hardness of the hardest pencil that does not lead to a visible scratch on the surface of the coating; the second value is the hardness of the softest pencil that leads to a visible scratch on the surface of the coating. Approximate layer thickness of the Ormocomp coating without moth eye structures is 15.4 µm; the moth eye-structured Ormocomp coatings have a residual layer thickness of about 15 µm. The results are displayed in As displayed in in triplo. The values in Accelerated ageing tests are performed on PMMA samples coated with non-structured and moth eye-structured Ormocomp to evaluate their durability. To get a first impression of the durability, three commonly applied accelerated ageing tests were performed: the damp heat test (1000 h at 85 °C and 85% relative humidity), the thermal cycling test (200 cycles between −40 °C and 85 °C) and the humidity freeze test (10 cycles between 85 °C and 85% relative humidity and −40 °C). The results of these durability tests are shown in The adhesion of Ormocomp to PMMA is very good; no delamination is observed after the tests. As demonstrated by the results displayed in in casu droplet dispensing. The web and the resist are pressed onto the drum, which contains the moth eye-structured replica, by the first roller (imprint roller). In our specific case, the drum itself is not structured, but, instead, moth eye-structured PDMS replicas are fitted onto the drum. Hence, the resulting moth eye-structured ARC is not seamless. The resist is cured by high-powered UV LEDs, using the web tension to make sure the resist is cured in contact with the structure. After that, the release of the cured resist from the drum is facilitated by the second pressure roller, the delamination roller.To apply the above mentioned ARC to large area, high-volume applications, it is necessary to use a continuous coating and structuring process. For that purpose, we designed process equipment to apply and structure coatings on foil in a roll-2-roll (R2R) fashion. For a schematic representation and pictures of the R2R pilot equipment, see −1. The web width is 30 cm; the drum has a circumference of 60 cm. A UV blocking vented cover prevents any harmful gases from escaping to the environment  and delamination roller pressure (roller 2). The tool is able to run at speeds in between zero, one and 3 m·minment see .The tool is fitted with a droplet dispensing system. The thickness of the resist can be controlled by changing the pressure of the first roller, but is highly dependent on the viscosity of the resist. The tool is fitted with a high-power LED bar to cure the resist through the foil. The wavelength of the UV LEDs is 395 nm.n = 1.57). An SEM image of the moth eye-structured coating prepared at a line speed of two meters per minute using the above-mentioned R2R setup is displayed in In the experimental set-up for producing moth eye-structured Ormocomp layers, we used identical h-PDMS stamps as used in the wafer-by-wafer experiments. The stamp was mounted on the imprint drum. The resist was applied to the web using the above-mentioned droplet dispensing system. After optimization of the process parameters, we were able to produce moth eye-structured Ormocomp coatings on polyethylene terephthalate .The HT-AR-09 mold was supplied by NIL Technology. This nickel mold contained moth eye nanostructures with a 250 nm pitch, 300 nm average height, a peak-to-peak distance of 300 nm and was supplied with an anti-sticking layer.The moth eye nanostructures were applied through NIL. The process used consisted of two main steps: preparation of the replica and structuring of the Ormocomp resist, as illustrated in Nanostructure replication into a thin and flexible, UV-transparent silicone polymer was performed using the Holotools HT-AR-09 mold and a stack of two layers of polydimethylsiloxane (PDMS). A first layer of hard PDMS (h-PDMS) was applied on top of the mold. This material has a higher e-modulus compared to regular PDMS, but a much lower elongation at break. The material is quite brittle and requires a bilayer approach, using a second layer of softer PDMS .The h-PDMS mixture was prepared by subsequently adding 30 mg of 1,3,5,7-Tetravinyl-1,3,5,7-tetramethylcyclotetrasiloxane, 6.8 g vinylmethylsiloxane-dimethylsiloxane trimethylsiloxy terminated copolymer, 2 g (25%–35% methylhydrosiloxane) of dimethylsiloxane copolymer and 30 mg of 1,3,5,7-Tetravinyl-1,3,5,7-tetramethylcyclotetrasiloxane. All four components were supplied by ABCR and used without purification. The materials were mixed using a speedmixer at 3500 rpm for 60 s. The h-PDMS was spin coated on top of the mold within 15 min after preparation, at 1000 rpm for 40 s. It was partly cured on a hotplate at 60 °C for 2 min.Sylgard 184, supplied by Dow Corning, was used as the soft PDMS material as supplied. It was mixed in a mass ratio of part A:B 10:1 in a speedmixer at 2000 rpm for 60 s. The material was casted on top of the partly cured h-PDMS, until the mold was completely covered with a PDMS layer of approximately 0.5 mm. The stack was cured in a convection oven at 80 °C for 5 h. The replica was peeled off the mold and used in the NIL process.The UV-curable material used for imprinting was Ormocomp, supplied by Micro Resist Technology. It has a 100% solid content and was used as delivered by the supplier.Prior to the wet film application, the substrates were dried in a convection oven for 10 min. The oven temperature for PMMA was 80 °C; for PC, 120 °C.Ormocomp was applied with a 20 µm Bird applicator and prebaked in a convection oven at 80 °C for 15 min. The step-and-flash NIL process was performed using the Nanoimprint Technology Platform from NIL Technology. After the post-exposure bake in a convection oven at 80 °C for 15 min, the PDMS replica was removed from the sample.The layer thickness was measured using a standard profilometer.The images were made with the Zeiss Orion Plus HIM. This microscope is located on a special vibrational-free floor and placed in an in-house developed acoustic enclosure. The samples were scanned point by point with a sub-nanometer spot, with an acceleration voltage of 25 kV and a typical beam current between 0.1 and 1 pA. The images were recorded with an ET detector, collecting an amount of secondary electrons generated by beam sample interaction. A 5 nm carbon layer was deposited on the samples prior to analysis.A Nanosurf Flex AFM with high aspect ratio tips was used in tapping mode for AFM measurements.A Shimadzu UV3600 spectrophotometer was used for total reflection and transmission measurements with an integrating sphere. The incident angle was 7°; the wavelength range was 300 to 1000 nm. The sampling interval was 5.0 nm and slit width, 20 nm.A. Since the force, F, is determined by the weight of the carrier and, therefore, constant, the contact pressure, p, changes gradually from low pressure (soft pencils) to high pressure (hard pencils).Pencil hardness tests were performed according to ISO15184:1998. In all cases, three substrate samples were coated with Ormocomp resist, moth eye-structured using NIL and cured. 24 h after preparation, the pencil hardness of the coating was determined on three different positions per sample. Ergo, all values for pencil hardness are average values of nine measurements. In the pencil hardness test, pencils of different hardness ranging from 9B (softest) to 9H (hardest) are moved over a coating surface using a well-defined carrier to guarantee constant weight. Under the applied weight, the soft pencil tips deform more than the harder ones, leading to a larger contact area, in triplo.Three accelerated lifetime tests were performed on PMMA substrates with moth eye-structured Ormocomp, with the non-structured coating and with blank PMMA substrates. Reflection VIS was measured before and after testing. All samples were prepared and tested Three commonly applied accelerated ageing tests were performed: the damp heat test (1000 h at 85 °C and 85% relative humidity), the thermal cycling test (200 cycles between −40 °C and 85 °C) and the humidity freeze test (10 cycles between 85 °C and 85% relative humidity and −40 °C).i.e., the lack of suitable coating materials and a process for large area, high-volume applications—can be eliminated to a large extent, paving the way for cost-efficient and large-scale production of durable nanostructured moth eye-type ARCs on polymer substrates.In conclusion, we prepared moth eye-structured Ormocomp coatings on PMMA and PC, using a wafer-by-wafer step-and-flash NIL process. The reduction in VIS reflection achieved with these coatings was 3.5% and 4.0%, respectively. The adhesion of the coating to both substrates was good. The moth eye-structured coating on PMMA demonstrated good performance in three prototypical accelerated ageing tests: the damp-heat, thermal cycling and humidity-freeze test. The pencil hardness of the moth eye-structured Ormocomp coatings on both substrates was <4B, which is less than required for most applications and needs further optimization. In addition, we developed a R2R UV NIL pilot scale process and produced moth eye-structured Ormocomp coatings on PET at line speeds up to two meters per minute. The resulting coatings showed a good replication of the moth eye structures and, consequently, a lowering in reflection of the coated PET of 3.0%. Further optimization of the process is currently ongoing in our laboratories. Ergo, we successfully demonstrated that the two main disadvantages of moth eye-structured graded index ARCs—


Helicobacter pylori infection is considered as an excellent model of chronic inflammation-induced tumor development. Our project focuses on gastric MALT lymphoma (GML) related to H. pylori infection and mediated by the chronic inflammatory process initiated by the infection. Recently, microRNAs (miRNAs) have emerged as a new class of gene regulators, which play key roles in inflammation and carcinogenesis acting as oncogenes or tumor suppressors. Their precise characterization in the development of inflammation and their contribution in regulating host cells responses to infection by H. pylori have been little explored. Our goal was to analyze the changes in miRNAs in a GML mouse model using BALB/c mice thymectomized at day 3 post-birth (d3Tx model) and to clarify their implication in GML pathogenesis. PCR array followed by RT-qPCR identified five miRNAs  overexpressed in the stomachs of GML-developing d3Tx mice infected by H. pylori. The analysis of their putative targets allowed us to identify TP53INP1, an anti-proliferative and pro-apoptotic protein, as a common target of 4 of the 5 up-regulated miRNAs. We postulate that these miRNAs may act in synergy to promote the development of GML. miR-142a was also overexpressed in mouse sera samples and therefore could serve as a diagnostic marker. In situ hybridization on gastric samples with miR-142a revealed a global up-regulation of this miRNA by the tumor microenvironment at the lymphoma stage. Dysregulation of miR-21a, miR-135b, miR-142a, miR-150, miR-155 could play a critical role in the pathogenesis of GML and might offer potential applications as therapeutic targets and novel biomarkers for this disease. Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa of about 50% of the world population. The infection causes an inflammation (gastritis), which is superficial and asymptomatic at first, but may evolve toward gastric or duodenal ulcer, gastric adenocarcinoma, or gastric mucosa-associated lymphoid tissue (MALT) lymphoma (GML)  (q21:q21)-negative GML cases . A large cohort of d3Tx BALB/c mice and non-thymectomized controls (NTx) were either infected with H. pylori or non-infected. GML-like lesions were only observed in the infected-d3Tx mice. In these animals, the gastric lamina propria was infiltrated by polymorphonuclear and lymphoid cells, the latter mainly organized in follicles composed of B cells with few infiltrating T cells. The monoclonality of infiltrating B cells was demonstrated and was strongly correlated with the presence of lymphoepithelial lesions. Using the material obtained in Chrisment et al.  (7 NI and 19 H. pylori infected mice). miRNAs were also extracted from paraffin-embedded gastric tissues using the miRNeasy FFPE kit (Qiagen). Two 20 μm sections or four 10 μm sections from 6 d3Tx mice stomachs (3 NI and 3 infected mice) were used for miRNA extraction according to the manufacturer's instructions. miRNAs were extracted from 500 μl of serum (diluted 4-fold), by adding 750 μl of TRIzol® LS Reagent  according to the manufacturer's instructions.miRNAs were extracted from frozen gastric biopsies using the miRNeasy mini kit  according to the manufacturer's instructions. miRNA samples were extracted from non-thymectomized (NTx)   were separated using SDS/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were incubated with the rat anti-mouse TP53INP1 monoclonal antibody . Secondary Polyclonal Rabbit Anti-Rat Immunoglobulins/HRP antibody  was used to bind primary antibody and the reaction was detected by ECL Prime Western Blotting Detection Reagent . After blotting, the membranes were stained with SYPRO Ruby Protein Blot Stain (Invitrogen) and scanned with the Molecular Imager PharosFX (Bio-Rad) for the quantification of protein load. Specific antibody signals were normalized against total protein amount for each entire lane. The signal intensities were analyzed using ImageJ software.in situ hybridization (ISH) of miR-142a, deparaffinized, and rehydrated tissue sections of 6 μm thickness were digested by Proteinase K  for 20 min at 37°C, and post-fixed in 4% paraformaldehyde in PBS for 10 min at 4°C. After three washes in PBS, the tissues were prehybridized for 1 h at 55°C in 25% formamide, 4X SSC, dextran sulfate 200 mg/ml, and tRNA 220 μg/ml, and hybridized overnight at 55°C in the same buffer containing 100 nmol/l anti-miR-142a probe or 5 nmol/l anti-RNU6 probe labeled at both 3′ and 5′ ends with digoxigenin. After successive stringent washes  at 55°C, the slides were incubated with blocking solution  for 1 h at room temperature. An anti-digoxigenin antibody coupled to alkaline phosphatase  was used to reveal hybridization of the anti-miRNA probes. After 3 washes, a 2 h incubation step at room temperature in 1-StepNBT/BCIP plus Suppressor Solution  was performed. Slides were dehydrated and mounted with Eukit-mounting medium .For Slides were scanned using a digital slide scanner  equipped with a Zeiss objective  and a high-resolution color camera  available at the Experimental Histopathology Platform, US 005 UMS 3427-TBM CORE. The images were read using the Panoramic Viewer software version 1.15.4 (3DHISTECH Ltd).*p < 0.05).Statistical analyses were performed with GraphPad Prism 5.01 . The Mann-Whitney test was used as nonparametric test to compare the distributions of two unmatched groups. Differences were considered significant when p was inferior to 0.05 . miRNA expression from a pool of RNA of 2 infected d3Tx mice was compared to a pool of RNA from 2 NI d3Tx mice (2 of the 3 mice that were included in the first PCR array run). Among the 372 miRNAs represented on the array, 136 were undetectable or weakly expressed , leaving 236 miRNAs for the analysis. Among these, the expression of 19 miRNAs was noticeably increased at GML stage , whereas the expression of 14 miRNAs was decreased  revealed that 4 of the 5 miRNAs up-regulated in our model, namely miR-135b, miR-142a, miR-150, and miR155, have potential binding sites within the 3′ UTR of the mRNA of the pro-apoptotic gene TP53INP1 and hence would be able to down-regulate it.Identification of miRNA target genes is essential for determining miRNA function. Recent studies have indicated that a single miRNA may regulate more than 200 target genes. Furthermore, one target gene could be regulated by many miRNAs. The interrogation of the TargetScan database for predicting target genes  or lymphoid infiltration (lymphoid infiltration = 0). As expected, the production of TP53INP1 was decreased in GML mice relative to control mice Figure .miRNAs have been previously described as potential diagnostic markers of human diseases Cho, . RelativHence, with further confirmation, miR-142a has a potential to be a non-invasive diagnostic marker of gastric MALT lymphoma.In order to identify the miR-142a-producing cells, either lymphocytes or cells from the tumor microenvironment such as epithelial cells, ISH for miR-142a was performed on sections from NI and infected d3Tx mice stomachs and showed that it was, indeed expressed, by both lymphoid infiltrates and the tumor environment Figure .H. pylori-infected d3Tx mice, we showed an over-expression of miR-21a, miR-135b, miR-142a, miR-150, and miR-155 in mice stomachs at the lymphoma stage. Studying the predicted targets of these miRNAs allowed us to identify a common regulatory target, which corresponds to the TP53INP1 pro-apoptotic factor. Our work highlighted that miR-21a is a marker of intensity of gastric inflammatory scores and for the first time, indicated that miR-142a could be a non-invasive marker of GML.miRNAs play an important role in the regulation of the immune system  mRNA. TP53INP1 is a protein that is over-expressed during stress responses including inflammation. It is a proapoptotic stress-induced p53 target gene. p53 activates TP53INP1 transcription, and overexpression of TP53INP1 induces cell cycle arrest and apoptosis  or a tumor suppressor , thus making the mechanisms involved in lymphomagenesis more complex. The miRNAs over-expressed in our study could act synergistically by targeting multiple signaling pathways Figure . On the H. pylori and direct for EBV) could be linked through common miRNA deregulation disrupting the same signaling pathways.Interestingly, an over-expression of miR-21a and miR-155 has also been described in NK/T cell proliferation induced by Epstein Bar Virus (EBV). These miRNAs are involved in immortalization of lymphocytes  are up-regulated in gastric lymphomagenesis in mice, were identified in our study. Upregulation of miR135b was never described before in GML cases and neither was the upregulation of miR-142a in mice sera. In addition, this study is in agreement with the previous findings obtained using mouse or human materials describing the upregulation of miR-142a and miR-155 at GML stage . Finally, further analysis of these miRNAs in human biopsies or histological sections would validate their deregulation at the GML stage and define potential new therapeutic targets. miR-142a could also be a potential marker of GML.PF: Wrote the manuscript, performed the experiments, analyzed the data. CC: Performed the experiments, analyzed the data. CS: Wrote the manuscript, analyzed the data. JI: performed the experiments. ES: Performed the experiments. AL: Wrote the manuscript. AG: Performed the experiments. VK: Wrote the manuscript. PD: Analyzed the results. FM: Analyzed the results. PL: Designed and analyzed the experiments, wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide–hydroquinone–horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out. Due to their central role in inflammatory-based diseases, cytokines are considered as potential biomarkers for cancer, asthma, and several other diseases . The cytEspecially electrochemical immunosensors could be suitable, as they have the potential to be integrated into a fluidic system and can be automated ,7,8. TheUp to now, research has focused primarily on sensor development rather than of system development. One example of an electrochemical immunosensor system, developed by Schuhmacher et al., comprises a lab-on-chip system for multi-parameter analysis with optical and electrochemical read-out . FurtherMost immunosensors use enzymes bound to the detection antibodies for signal generation and amplification, but hardly any electrochemical immunosensor or system pays attention to the temperature or pH optimum of the enzyme. Besides enzyme kinetics depending on temperature, temperature influences the rate of the electrode reaction and affects transport processes of involved chemical substances to the electrode surface ,21. In tHence, the aim of this work was to develop an electrochemical immunosensor and to integrate it into a thermostat-controlled fluidic system for read-out. Accordingly, two sets of results are reported here. First, an electrochemical immunosensor with an enzyme for read-out was developed to detect IL-13 as a model protein of the cytokine family. Second, a fluidic system with temperature control realized by Peltier elements was built up to enable a temperature-controlled read-out at the temperature optimum of the sensors enzymatic label. For this purpose, the immunosensor was prepared in batch and amperometric read-out was performed in a temperature-controlled fluidic system to evaluate the influence of the temperature on the enzyme activity and thus on the electrochemical sensor response. Under operation at the temperature optimum of the enzyme, an enhanced sensitivity of the electrochemical immunosensor was assumed. Finally, the immunosensor selectivity was evaluated in a complex biological matrix.The sensor architecture and signal generation are depicted in N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (EDC), bovine serum albumin (BSA), Casein, Tween 20, hydrogen peroxide 30%, Triton X-100, Halt Protease & Phosphatase EDTA-free Inhibitor Cocktail, QuantiPro BCA Assay Kit, and hydroquinone were obtained from Sigma-Aldrich . PBS pH 7.2 (Gibco), RPMI-1640 medium, and foetal bovine serum (FBS) were obtained from Thermo Fisher Scientific . Sodium chloride was obtained from Merck . 2-(N-Morpholino)-ethanesulfonic acid (MES) was purchased from Amresco . N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) was obtained from Alfa Aesar . Immunoreagents were from the human IL-13 DuoSet ELISA Kit obtained from Bio-Techne . For the immunosensor, a streptavidin–horseradish peroxidase (HRP) conjugate (ABIN964537) from antibodies-online was applied .Disodium hydrogen phosphate, sodium phosphate dibasic, tris-(hydroxymethyl)-aminomethane (TRIS), Screen-printed electrodes (SPE) with carboxyl-functionalized single-walled carbon nanotubes (DRP-110SWCNT) as working electrode (d = 4 mm) were purchased from Dropsens . The counter and reference electrode on the sensor strip are made of carbon and silver, respectively. All potentials are given vs. the reference electrode on the sensor strip. For electrochemical experiments, a PalmSens3 potentiostat/galvanostat with the appropriate Software PSTrace 5.1 was obtained from PalmSens .2O2. For substrate conversion, 20 min was given, avoiding direct light. Finally, the reaction was stopped by the addition of 50 μL of 2 M sulfuric acid. Read-out was done at 450 nm and at 540 nm with a microplate reader SpectraMax® M3 from Molecular Devices . Values at 540 nm were subtracted from values at 450 nm to correct for optical imperfections of the plate.Before immunosensor preparation, the human IL-13 DuoSet ELISA Kit (Bio-Techne) was used to conduct an ELISA with optical read-out as reference according to the manufacturer’s protocol, which is briefly described as follows. All steps of the procedure were performed at room temperature. Per well, 100 μL of capture antibody were incubated overnight, followed by washing and blocking with BSA solution for 1 h. After washing, 100 μL of IL-13 standard solution was added per well and incubated for 2 h. Subsequently, washing was performed and incubation with 100 μL detection antibody per well for additional 2 h. Once washed, 100 μL of the streptavidin–HRP conjugate was added per well and incubated for 20 min. Then, washing was performed followed by the addition of 100 μL recommended substrate solution, per well, containing 3,3′,5,5′-Tetramethylbenzidine (TMB) and H®. Before continuing with the immunosensor set-up, a successful immobilization of capture antibodies was verified. Specially, for this purpose, prepared electrodes with capture antibodies were applied. To proof the successful immobilization of capture antibodies, a species-specific antibody labelled with HRP against the capture antibody (ABIN101754 from antibodies-online) was used. Amperometric measurements of the electrodes were performed according to 2.6.The immunosensor preparation was accomplished in an in-house designed chamber of polyether ether ketone (PEEK), with all steps performed at room temperature. The chamber was blocked before use with 0.1 M PBS pH 7.4 plus 1% (w/v) BSA for 15 min. The 0.1 M PBS solution was composed of disodium hydrogen phosphate, sodium phosphate dibasic, and sodium chloride, modified according to . The immFor further immunosensor set-up, electrodes were washed with 100 μL of 0.1 M PBS pH 7.4 to remove residual storage buffer. Then, the electrodes were blocked with 100 μL of a 2% (v/v) Casein in 0.1 M PBS pH 7.4 for 20 min prepared from a 5% (w/v) Casein stock solution, compare , to prevx and the inserted value for y in μA was the sum of the mean of the blank and three times the standard deviation of the blank [Amperometric measurements were conducted in a beaker containing 10 mL of 1 mM hydroquinone in 0.1 M PBS pH 6 under magnetic stirring. The applied working potential was 0.0 V, which was determined as described in the he blank . Here, tEquation of four-parameter logistic model:The measurements in the beaker were transferred to a flow cell system with minor modifications: Amperometric measurements in the flow cell were conducted by applying a continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6 with a flow rate of 0.8 mL/min. The applied potential was 0.0 V. To detect, 100 μL of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6 were added with a flow rate of 0.4 mL/min to reach a final concentration of 0.5 mM hydrogen peroxide when diluted in the continuous flow. The obtained peak signal was recorded until the baseline was reached again. For data analysis, the last 100 s of the baseline were averaged and subtracted from the peak current. This was repeated for three different immunosensors prepared in the same manner (triplicates), unless otherwise stated. The mean current change and the standard deviation were calculated from these three results. Finally, signals were fitted with the four-parameter logistic model as described above.® 2016 from Dassault Systemes  and was composed of poly-(methyl methacrylate) (PMMA) (The flow cell was designed with the CAD Software Solidworks) (PMMA) . To realg and 4 °C for 30 min. Total protein concentration was determined by applying the QuantiPro BCA Assay Kit . Lysates were stored until use at −80 °C. For selectivity determination, IL-13 immunosensors were prepared according to To evaluate the selectivity of the IL-13 immunosensor, cell lysate of human lung cancer cell line NCI-H1975 was prepared. Cells were cultured in RPMI-1640 medium with 10% (v/v) FBS and kindly provided by a colleague. For lysate preparation the cells were rinsed two times with PBS pH 7.2 (Gibco by Life Technologies) and lysed by adding 1 mL of lysis buffer  Triton X-100, 1x Halt Protease & Phosphatase EDTA-free Inhibitor Cocktail), compare , to eachPrior to the development of an IL-13 immunosensor, the immunoreagents form the ELISA kit were checked by performing an ELISA according to the manufacturer’s protocol (2.4). The calibration curve obtained is in line with the manufacturer’s specifications and a LOD of 99 pg/mL was calculated . The assThe electrochemical IL-13 immunosensor was set up as described above . For sen2O2 vs. hydroquinone and H2O2) and the read-out methods . Hydroquinone was chosen over TMB for the sensor to avoid passivation and poising caused by oxidized TMB [For the IL-13 immunosensor, a LOD of 7.0 ng/mL of IL-13 was calculated. Hence, the here-developed immunosensor is 70-fold less sensitive compared to the conventionally performed ELISA (3.1). Moreover, the sensor showed a small dynamic range from 7.0 ng/mL to 20 ng/mL of IL-13, which is at the upper end of the dynamic range of the ELISA  nanoparticles on graphene oxide sheets for cancer biomarker detection [Compared to other electrochemical immunosensors, the developed IL-13 sensor is also less sensitive. One reason could be missing signal amplification. Other groups using poly-HRP conjugates for cytokine detection  or advanetection  reach deIn summary, the here-developed IL-13 immunosensor is less sensitive compared to ELISA and to other electrochemical immunosensors. With the aim to enhance the sensitivity of the sensor, a temperature-controlled read-out was introduced. For this purpose, a fluidic system with temperature control was developed. Further, the fluidic system is suitable to minimize manual interaction. Results are described below.2O2 proved that these peaks are not caused by the injection event itself .The signal increase with higher temperature reflects the higher activity of the enzymatic label HRP. Higher signals could be advantageous to improve the detection limit. Besides enhanced enzyme activity, temperature increase supports mass transfer and electron transfer, which may contribute to the observed offset. Further, temperature control in general may be convenient to reduce variations of the sensor response as described in literature . UnsatisIn a first selectivity study, the ability of the developed immunosensor to detect IL-13 concentrations in a complex biological matrix was assessed. Therefore, cell lysate of human lung cancer cell line NCI-H1975, spiked with defined IL-13 concentrations (0–15 ng/mL) was analyzed. For comparison, buffer containing equal IL-13 concentrations was tested. Lysate and immunosensor preparation are described in The here-presented approach addresses a temperature-optimized read-out of an amperometric IL-13 immunosensor in a fluidic system for the first time. With a temperature optimum at 30 °C for read-out, a clear improvement of sensor performance by temperature control could be demonstrated, although the amperometric immunosensor system, with a LOD of 5.4 ng/mL of IL-13 at 30 °C, showed to be 55-fold less sensitive compared to the ELISA. This may be due to differences in the two systems as described. Hence, the IL-13 sensor system could be improved with respect to background signal, noise reduction, and signal amplification. Additionally, the quality of the used SPE should be investigated and the sensor preparation procedure needs to be improved in future work. To apply the sensor in clinics, further selectivity studies as well as clinical studies have to be performed. Although further improvements are to be done, the sensor system is a first step towards an automated immunosensor platform.


Normal human adipose-derived stem cells  were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3--2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu Adipose tissue is an excellent source of adult stem cells, which are called adipose-derived stem cells (ADSC) . They hoAntibiotics are extremely important in transplantation and regenerative medicine procedures. Isolation and cultivation of adipose-derived stem cells usually involves the presence of the penicillin-streptomycin mixture ,5,8,9 al2+), which was reported to be less toxic to animal cells and more potent in combating fungal infections [2+ in stem cell culture may favorably affect their ability to proliferate and differentiate.One of the most effective antifungal compounds is amphotericin B (AmB), a polyene antimycotic drug that belongs to the macrolides ,15. Due fections ,17,18,192+ on stem cells, especially related to their impact on ADSC, has not been studied so far. The object of the present study was to evaluate the influence of these antibiotics on the growth and differentiation potential of ADSC. In the first step the influence of antibiotics and their combinations on stem cells viability was tested. Next, the potential of adipogenesis and osteogenesis promotion by antibiotics in the absence of dedicated differentiation factors was assessed (in basic medium). Obtained results have prompted us to the evaluation of the influence of antibiotics and their combinations on differentiation into osteoblasts in the dedicated osteogenesis medium.The influence of penicillin, streptomycin, amphotericin B and AmB-Cu2+, PS-AmB and PS-AmB-Cu2+ showed a statistically significant decrease of viability comparing to the control  . The viability of cells was assessed indirectly by the measurement of total cellular protein content in ADSC after 24, 48 and 72 h of incubation with antibiotics. Cells treated for 24 h with AmB, AmB-Cuctively) A. Moreov2+ showed higher viability than the control (p = 0.0004), AmB (p = 0.0001), PS- (p = 0.0004), PS-AmB- (p = 0.0371) and PS-AmB-Cu2+-treated cells (p = 0.0001) (p = 0.0082) and PS-AmB-Cu2+ (p = 0.0280).After 48 h, cells treated with AmB-Cu 0.0001) B. PS-AmB2+ and PS-AmB-Cu2+, comparing to the control , PS , and AmB groups  (p = 0.0104)Seventy-two hours of incubation caused a significant decrease in the viability of cells exposed to AmB-Cu 0.0002) C. AmB tr2+ treatment caused a statistically significant increase of mitochondrial oxidative activity comparing to the control (p = 0.0200), as well as cells exposed to AmB (p = 0.0049) and PS-AmB-Cu2+ (p = 0.0034) (Based on the results pertaining cell viability presented in  0.0034) A. 2+ and PS-AmB in comparison to control , PS  and AmB group  . 48 h exposure caused significant increase of mitochondrial activity of cells treated with AmB-Cu 0.0001) B. Moreov2+-, and PS-AmB-Cu2+-treated cells  (2+ comparing to the control and cells exposed to PS (p < 0.0030 in both comparisons)  C. Viabilarisons) C. Besides direct effects on cell viability, antibiotics may also affect the stemness phenotype of ADSC. Hence, we have assessed how the tested combinations of antibiotics may affect the expression of mesenchymal stem cell markers like CD73, CD90, and CD105, both at mRNA and protein level. CD90 mRNA in comparison to control. AmB caused significantly lower mRNA expression of CD90 marker compared to PS (p = 0.0086). None of the tested drugs or their combinations has changed the expression of CD73 mRNA level comparing to control, PS-, AmB-Cu2+-, and PS-AmB-treated cells   . CD105 mRNA expression was statistically significantly higher after treatment with PS comparing to control, AmB-, AmB-Cu2+-, PS-AmB- and PS-AmB-Cu2+-treated cells . AmB caused decrease of CD105 mRNA level comparing PS-AmB-Cu2+ (p = 0.0100). Fluorescence-activated cell sorting (FACS) analysis showed the presence of CD73, CD90 and CD105 markers at protein level in all analyzed groups. The percentage of marker-positive cells was slightly higher after treatment with antibiotics .We also analyzed the morphology of the adipose-derived stem cells after 14 days . We did HOXC8 . In AmB-treated cells, the expression of this marker was significantly lower comparing to PS, AmB-Cu2+ and PS-AmB-Cu2+ . The expression of LEP mRNA was significantly higher in cells exposed to PS in comparison to control, AmB-, AmB-Cu2+-, and PS-AmB-treated cells . Additionally, in cells treated with AmB, the LEP mRNA level was significantly lower than in PS-AmB-Cu2+-treated cells (p = 0.0053).The level of 2+ (2+-treated cells (OD = 0.65). The Oil Red O showed very weak staining of lipids in control cells and slightly stronger staining in cells treated with PS-AmB-Cu2+ . The optBGLAP, RUNX2, ALP and SPP1 mRNAs) by RT-qPCR. The ADSC were cultured for 14 days in standard Dulbecco’s Modified Eagle Medium (DMEM) without dedicated factors stimulating osteoblastic differentiation. The changes in the expression of osteoblast markers BGLAP and RUNX2 were similar in studied groups . AmB caused downregulation of this marker in comparison to PS-, AmB-Cu2+- and PS-AmB-treated cells . The expression of BGLAP mRNA level was higher in PS-treated cells comparing to control (p = 0.0079). After AmB treatment, the expression of BGLAP was lower in comparison to groups: PS, PS-AmB and PS-AmB-Cu2+ . 2+, PS and PS-AmB (2+ and PS-AmB-Cu2+ groups (p = 0.0380).Alizarin Red staining showed slight increase of extracellular calcium in the groups of cells treated with AmB-Cud PS-AmB . HoweverL-ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate) and combinations of tested antibiotics  and in AmB-Cu2+- (p = 0.0091) compared to PS-treated cells.Expression of RUNX2 was significantly lower in cells treated with AmB and AmB-Cu2+ compared to the control (p = 0.0019 and p = 0.0002) and PS-treated cells (p = 0.0134 and p = 0.0016). Moreover, the RUNX2 expression in AmB-Cu2+ was significantly lower than in cells treated with PS-AmB-Cu2+ (p = 0.0154).The mRNA level of SPP1 mRNA showed a statistically significantly lower expression in groups: AmB (p = 0.0002), AmB-Cu2+ (p = 0.0033) and PS-AmB (p = 0.0334), compared to control. There was also a statistically significantly lower expression of this marker in AmB- (p = 0.0005226) and AmB-Cu2+ groups (p = 0.008530) in comparison to PS-AmB-Cu2+-treated cells. ALP in the examined groups in comparison to control cells. The expression of ALP was significantly higher in AmB-treated cells compared to group PS (p = 0.0123). No statistically significant difference was observed in the expression of 2+ compared to control (p = 0.0001 and p = 0.0002). Calcium level was significantly higher for cells treated with AmB in comparison to control cells (p = 0.0015) and cells treated with PS (p = 0.0001), PS-AmB (p = 0.0030), and PS-AmB-Cu2+ (p = 0.0001). AmB-Cu2+ and PS-AmB caused increase of calcium content in comparison to PS- (p = 0.0001 and p = 0.0001) and PS-AmB-Cu2+-treated cells (p = 0.0001 and p = 0.0007).For osteogenesis, the presence of extracellular calcium was confirmed by Alizarin Red staining. As shown in Mesenchymal stem cells (MSC) are multipotent, somatic cells with multilineage differentiation ability. Furthermore, they also have immunomodulatory, angiogenic and antiapoptotic activities through the secretion of growth factors, cytokines and peptides. MSC can be isolated from bone marrow, skin, whole blood or adipose tissue. ADSC have many advantages such as a low risk of side effects when being harvested and the fact that the tissue is easy to obtain from almost every patient, or from medical waste upon cosmetic surgeries; hence there are no ethical issues. Moreover, the cells are easy to culture and they can differentiate into adipocytes, chondrocytes, osteoblasts and myocytes ,5,6,8,20Many endogenous and exogenous factors may affect the proliferation and differentiation ability of mesenchymal stem cells, e.g., derivation of adipose tissue, age of donors, comorbidities, method used to isolate the stem cells, as well as the duration and conditions of culturing them ,21. The Moreover, all methods of nonisogenic transplantation require the use of immunosuppressive agents, which induces a temporary immune deficiency, often facilitating dangerous fungal infections. The types of antifungal drugs that are used depend on an individual’s symptoms, concurrent illnesses and the species of fungi ,22. The 2+ on human proximal tubular epithelial cells (RPTEC) and fibroblasts encouraged us to assess also its influence on mesenchymal stem cells [To gain better understanding of the used experimental systems in regenerative medicine and tissue engineering, we have evaluated the effect of antibiotics on the growth and differentiation of the MSC derived from the adipose tissue commonly used in cell culture settings and in the clinic. Additionally, previous studies reporting lower toxicity of AmB-Cuem cells ,18.2+ did not inhibit the growth of RPTECs [2+. After 48 h, we observed similar results in both assays (cell growth SRB—sulforhodamine B and MTT), and the highest proliferation of the cells occurred in the group that was treated with AmB-Cu2+. After 72 h however, an inhibition of protein production and mitochondrial activity was detected in the cells after exposure to AmB-Cu2+ compared to AmB alone. As the first days of the experiment caused an increased proliferation, the decrease observed after 72 h may be the result of the rapid depletion of nutrients in the culture medium of cell treated with AmB-Cu2+. In turn, observed increase of cells viability after 72 h of incubation with AmB may indicate that MSC inactivated this drug only after two days. Thus, we conclude that MSC tolerate the exposure to AmB-Cu2+ much better than AmB. Our finding in these assays indicates that the cellular changes depend on the time of exposure and type, as well as combination of antibiotics used in the experiment. Some discrepancies observed between the read-outs from SRB and from the MTT assay reflect the fact that both assays measure different cellular parameters.Our experiments examined the short- and long-term effects of antibiotics and antimycotics. The concentrations of penicillin-streptomycin were determined based on literature data ,11. In af RPTECs . This cof RPTECs . Both anf RPTECs . We howeWe have also made some interesting observations regarding the effects of tested combinations of antibiotics on the maintenance of MSC phenotype, as assessed by the expression of various stemnesses and differentiation markers. ADSC were cultivated in standard medium without factors stimulating differentiation into adipocytes or osteocytes. We noticed a slight increase of population of cells coexpressing MSC markers under exposure to tested drugs. However, AmB caused a decrease of MSC markers at mRNA level. This may result from the influence of amphotericin B on mRNA stability. HOXC8 (homebox 8) and LEP (leptin) genes. These genes are characteristic for white adipose tissue cells [LEP and HOCX8, most significantly the penicillin-streptomycin. However, lipid staining with Oil Red O resulted in a weak signal, except for cells treated with PS-AmB-Cu2+. Hence, the addition of PS may be beneficial in supporting adipogenic differentiation in desirable medical settings. On the other hand, some studies have proved that PS may be cytotoxic to embryonic stem cells. Furthermore, Zur Nieden and colleagues have reported that penicillin G inhibited the differentiation of embryonic stem cells into osteocytes and chondrocytes [To assess potential adipogenic differentiation, we measured the expression (RT-qPCR) of ue cells . To somedrocytes . Thus, wRUNX2 and BGLAP mRNA level. We did not detect the mRNA of ALP and SPP1. However, the staining with Alizarin Red revealed the presence of extracellular calcium content, therefore we decided to evaluate the influence of tested drugs on osteogenesis in the presence of dedicated factors stimulating this process. We observed significant differences between amphotericin B and penicillin-streptomycin on the induction of osteogenesis. This study showed that amphotericin B and its new form with copper ions did produce significant effects of the osteogenesis, especially the lower expression of RUNX2, which is an early marker of osteogenesis and the osteopontin (SPP1) gene, which is a late marker of osteogenesis. Interestingly, we did observed a higher expression of the gene coding osteocalcin (BGLAP), which is a medium marker of osteogenesis. Hence, we can assume that amphotericin B and AmB-Cu2+ influence osteogenesis in the presence of osteogenesis-induction factors, including dexamethasone, β-glycerophosphate and L-ascorbic acid. The combination of AmB or AmB-Cu2+ with penicillin-streptomycin markedly modulated the expression of osteogenesis markers. Alizarin Red staining confirmed that AmB promoted osteogenesis, while addition of penicillin-streptomycin caused weaker signal. The mechanism of the action of streptomycin is connected to its binding to the 30S subunit of the bacterial ribosome. However, it also possesses some affinity to the ribosomes in eukaryotic cells. Relier and colleagues suggested that streptomycin may act on eukaryotic cells through the disruption of protein synthesis, by affecting mitochondrial activity, by binding to certain RNAs or by interfering with miRNAs action [All antibiotics, except AmB, caused an increase of s action . This in2 incubator . The ADSC that were used for the experiment were at second passage.Normal human adipose-derived stem cells (ADSC)  were routinely maintained in a DMEM medium , which was supplemented with fetal bovine serum , at 37 °C in a 5% CO2+) [2+ with penicillin streptomycin (PS-AmB-Cu2+). Control cells were cultured in a medium without antibiotics. The cells were treated with 0.5 µg/mL of amphotericin B (AmB) , 0.5 µg/mL of amphotericin B complex with copper ions (AmB-Cu2+) ,30, 10 µ3 cells per well. The cellular protein content in the cells after exposure to antibiotics was tested using a Sulforhodamine B (SRB)-based in vitro toxicology assay kit  and a Wallac 1420 VICTOR plate reader  according to the manufacturer’s instructions. The total cellular protein content in the cells was measured after 24 h, 48 h and 72 h of exposure.The cells were seeded into a 96-well culture plate at a density of 5 × 103 cells per well. MTT-assay was performed similarly as described recently [For the cytotoxicity assay, the cells were seeded into a 96-well culture plate at a density of 5 × 10recently . BrieflyTotal RNA was extracted from the cells using a TRIzol reagent  according to the manufacturer’s instructions. The nucleic acid concentration was determined using a MaestroNano Spectrophotometer . The expression mRNAs was detected using the real time RT-qPCR technique with SYBR Green chemistry  and an Opticon™ DNA Engine Continuous Fluorescence detector  as was previously described . All of Cells were cultured for 14 days in medium without any differentiation factors in the presence of tested antibiotics and their combinations. Control cells were cultured in the medium without antibiotics. Cells were collected to the RT-qPCR analysis and for flow cytometry analysis . R&D Systems Human Mesenchymal Stem Cell Marker Verification Multi-Color Flow Cytometry Kit  was used for mesenchymal stem cells markers identification: CD73-CFS, CD90-APC, CD105-PerCP. Cell fluorescence was measured immediately after staining , and data were analyzed using software  measured as the percentile of gated cells in fluorescent channels with activities of the corresponding isotype controls.After 14 days of culture, the Oil Red O staining and RT-qPCR analysis of HOX2 and LEP mRNAs was performed.Oil Red O staining is a microscopic visualization of the lipid droplets that are characteristic structures for adipocytes. For quantification of staining after washing out unbound dye and extraction with the isopropanol from cells, spectrophotometric analysis for quantitative assessment was carried out. The obtained absorbance value was calculated based on a calibration curve prepared with a series of Oil Red O solutions of known concentrations. The OD of supernatants was read at 405 nm in 96-well plate using a Perkin Elmer Wallac VICTOR2.BGLAP, RUNX2, ALP, SPP1 mRNAs) was performed.After 14 days of cell culture without any differentiation factors, Alizarin Red staining and RT-qPCR analysis  and fixed in 70% ethanol . Next, the monolayers were washed twice with dH2O and then Alizarin Red S was added. The plates were incubated at room temperature for 30 min with shaking. Stained cells were visualized using an Olympus IX81 microscope  and a DP70 camera  was used for the photographic documentation. To quantify the staining, the acetic acid  method was applied to recover the stain from calcium deposits. Stain solutions were adjusted to pH 4.2 with ammonium hydroxide and samples were read at 405 nm in a 96-well plate using a Perkin Elmer Wallac VICTOR2.Alizarin Red staining was used to evaluate calcium deposits in the culture by the cells. Alizarin Red at a concentration 40 mM was prepared in ddHL-ascorbic acid 2-phosphate, 107 M dexamethasone and 10 mM β-glycerophosphate . Cells were cultured in the above-described media with or without antibiotics for another 14 days. After 14 days, the cells were collected and the Alizarin S red staining and RT-qPCR analysis  was performed. Next, the cells were plated and grown until there was 80% of the confluent after which the medium was replaced with a fresh DMEM medium with fetal bovine serum (FBS) and 0.2 mM p < 0.05 for all of the statistical tests. Statistical analysis was performed using Statistica 13.0 software . The values are expressed as the means and standard deviation (SD) for normally distributed data. A one-way ANOVA test and a Tukey’s post hoc test were applied to evaluate any differences in the examined groups. For the non-normally distributed data, values were expressed as median value (Me) with the 25th and 75th quartiles and the Mann–Whitney U test was applied to assess differences in the expression of genes. Differences between the different groups were compared using Kruskal–Wallis test. The level of significance was set at 2+ as well as their combinations with PS affect the level of differentiation markers, however AmB-Cu2+ is better tolerated by stem cells. Antibiotics should probably only be used in special situations such as the isolation of a cell from a biopsy and at the early stages of a culture after which a standard culture should be done without this addition. Finally, it would be advised to choose the combination of antibiotics and antimycotics with caution, in a more targeted fashion, when propagating ADSC and possibly also other cell types. The findings are especially important given a rapid increase of experimental use of stem cells, in particular induced-pluripotent stem cells (iPS), and MSC (often ADSC), in the development of various regenerative medicine techniques [In summary, our results suggest that the presence of the antibiotics that are commonly used in cell cultures is not completely neutral to the phenotype of ADSC. Moreover, they modulate the differentiation process. Both AmB and AmB-Cuchniques ,34,35. Rchniques ,36,37,38